Thrombotic disease is a major cause of morbidity and mortality in the modern world. Acute myocardial infarction and ischemic stroke are the first and third causes of death and disability in Western societies. Occlusive thrombosis results in loss of blood flow to vital organs producing local oxygen deprivation, cell necrosis and loss of organ function. There are major benefits to the rapid destruction of a thrombus, resulting in early re-canalization: it prevents cell death, reduces infarct size, preserves organ function, and reduces early and late mortality. Thrombolytic therapy is now administered to more than 750,000 patients per year worldwide, while many times that number could potentially benefit from such treatment.
Thrombolytic agents now used in the lysis of occlusive blood clots are plasminogen activators. Several different plasminogen activators are currently available for immediate clinical use and several new generation plasminogen activators are the subject of clinical testing: tissue plasminogen activator, tPA, and its second generation successor TNK-tPA, RETEPLASE™ (a deletion mutant of tPA), single chain urokinase-type plasminogen activator (scuPA, or pro-urokinase), urokinase (UK), streptokinase (SK), and anisoylated plasminogen streptokinase activator complex (APSAC). tPA, scuPA, and UK are normally to be found at low levels in humans. Streptokinase is a bacterial enzyme with a powerful thrombolytic activity. APSAC is an anisolated streptokinase-plasminogen complex. In all cases the plasminogen activators are capable of converting the zymogen plasminogen to the active protease plasmin. The advantage offered by tPA and scuPA (and, to a lesser degree, APSAC) is that their activation of plasminogen is fibrin specific; binding to fibrin is a prerequisite for their full proteolytic activity to be realized (Haber et al., 1989). Urokinase and streptokinase can activate plasminogen in the absence of fibrin. Such variation in the affinity for fibrin has important consequences as to the extent to which systemic bleeding occurs in animal models; these differences, however, have not been appreciated clinically.
Plasminogen activators universally exert their thrombolytic potential by activating circulating zymogen plasminogen into plasmin. Plasmin, the principle fibrinolytic enzyme in mammals is a serine protease with trypsin-like specificity that is derived from the inactive zymogen precursor plasminogen circulating in plasma. Plasminogen itself is a 791 amino acid polypeptide having an N-terminus glutamate residue. Plasminogen activators such as tissue plasminogen activator (tPA) or urokinase will cleave the single-chain plasminogen molecule, to produce active plasmin, at the Arg561-Val562 peptide bond. The resulting two polypeptide chains of plasmin are held together by two interchain disulfide bridges. The light chain of 25 kDa carries the catalytic center and is homologous to trypsin and other serine proteases. The heavy chain (60 kDa) consists of five triple-loop kringle structures with highly similar amino acid sequences. Some of these kringles contain so-called lysine-binding sites that are responsible for plasminogen and plasmin interaction with fibrin, α2-antiplasmin or other proteins.
The inherent problem with the therapeutic use of existing plasminogen activators such as tPA, UK and SK is bleeding complications associated with their use, including, for example, gastrointenstinal hemorrhage in up to 20% of patients. Intracranial hemorrhage, which is clinically the most serious, is a frequent and lethal side effect of current thrombolytic therapy, and occurs in approximately 1% of patients. The mechanism for bleeding is multifactorial and is believed to be due to unmasking of a vascular injury by lysis of a protective hemostatic plug and consequent loss of vascular integrity. This is combined with the systemic activation of the fibrinolytic system and its attendant depletion of clotting factors. The focus of much recent research has been on generating modified plasminogen activators that exhibit improved fibrin specificity; this was expected to reduce the amount of bleeding complications. In some cases, these novel activators tend to preserve the circulating levels of such clotting factors such as fibrinogen, Factors VIII and V, plasminogen, and α2-antiplasmin. They specifically target and bind to the fibrin molecules that reside in a thrombus, and will only act upon plasminogen when so bound. This has the result that plasminogen is cleaved to the active protease plasmin only in the vicinity of the thrombosis and the level of non-specific systemic cleavage of fibrin is reduced. However, the number of bleeding complications with these new plasminogen activators remains significant.
The clinical success for thrombolytic drugs such as tissue plasminogen activator (tPA), streptokinase and urokinase in reducing the extent of a thrombotic occlusion of a vascular vessel is established. Plasminogen activators have therefore become a treatment of choice in the management of acute myocardial infarction and some other thrombotic conditions. Nevertheless, various disorders, including myocardial infarction, occlusive stroke, deep venous thrombosis and peripheral arterial disease, remain a serious clinical problem and the known plasminogen activators currently used suffer from several limitations that impact their overall usefulness in the elimination of a thrombus. In myocardial infarction, vascular flow is restored within 90 minutes in approximately 50% of patients, while acute coronary re-occlusion occurs in roughly 10% of patients. Coronary recanalization requires on average 45 minutes or more. Residual mortality, principally due to intracerebral hemorrhage, is still at least 50% of the mortality level in the absence of thrombolysis treatment.
Most research in the area of thrombolytics has focused on improving the efficacy and fibrin specificity of existing plasminogen activators as well as finding new ones. Much of this effort has concentrated on targeting the plasminogen activators to the fibrin that forms the scaffold of a thrombus and to improve the pharmacokinetics of the activators when administered into the blood stream. This would allow their administration as bolus doses rather than as a continuous delivery that prolongs exposure of the patient to the active agent, and the accompanying risk of undesirable systemic hemorrhage.
Based on the results of Phase II clinical trials with targeted plasminogen activators such as TNK-tPA, vampire bat salivary plasminogen activator, however, the anticipated improvement in safety profiles of the new plasminogen activators have not been realized clinically following thrombolytic therapy. The percentage of moderate and major bleeding episodes, including intracranial hemorrhage and stroke, were comparable with the original unmodified tPA. The clogged arteries were not opened earlier, and the rate of re-occlusions remained unchanged. It appeared that the only benefit these activators have is the prolonged plasma half-life and the possibility of bolus administration.
Another problem with plasminogen activators is that they have limited efficacy in the treatment of long clots found in peripheral arterial occlusions (PAO). These thrombi are typically aged and can grow to a significant size. The average size of peripheral thrombi found both in the native arteries and grafts is 31±11 cm. Aged and retracted thrombi are deficient in plasminogen, which therefore limits the susceptibility of old thrombi to plasminogen activator-induced thrombolysis. It is quite common for a patient with a PAO to be treated for 24 hours or more with urokinase and even after this prolonged period not to have complete patency of the vessel. The problem is greater with the delivery of the existing thrombolytic agents via catheter directly into the interior of the thrombus where there are reduced levels of the plasminogen substrate.
Further, thrombotic occlusion of hemodialysis grafts (hemodialysis graft occlusion—HGO) is a significant problem affecting those who must undergo regular hemodialysis treatment. More than 250,000 grafts are installed each year in the United States alone. Each graft lasts only about one year, and there are only three available positions where grafts can be placed in the human patient. Arteriovenous (A-V) grafts are used every three days for the dialysis of patients. Their effective use is quite often a matter of life and death for such patients.
HGOs occur regularly and must be treated prior to subsequent use of the grafts. Three modalities of treatment are currently used: pharmacologic, mechanical, and pharmaco-mechanical. Current pharmacological agents used are plasminogen activators, e.g., tissue plasminogen activator or tPA. These agents generate plasmin from endogenous plasminogen to degrade the occluding thrombi. Mechanical means for disruption of occlusions include use of crossed pulse-spray catheters (historically used with urokinase, heparin, etc., as the “pharmaco-” component in pharmacomechanical disruption) or fragmentation using a balloon catheter (Gibbens, D. T., et al., Tech. Vasc. Interv. Radiol., 4(2):122-126 (2001); Valji, K. et al., Am. J. Roentgenol., 164(6): 1495-1500 (1995)), and a percutaneous thrombolytic device (Trerotola, S. O., et al. Radiology, 206(2)403-414 (1998)). None of the pharmacological treatments are currently approved for HGO treatment (they are used “off-label” only).
Mechanical methods may not satisfactorily remove or fully disrupt thrombi. Similarly, pharmacological methods that depend on plasminogen activators may be limited in fibrinolytic activity by the amount of endogenous plasminogen available within the graft. This circumstance can complicate alleviation of HGO because the large clots (4-6 grams) characteristic of HGO occur within a relatively small volume of graft (10-12 ml). Additionally, as a volume of solution containing plasminogen activators is infused into the thrombosed catheter or graft, the serum within the device is displaced—further removing any available plasminogen for activation. In addition, clots that make up HGOs can be more difficult to dissolve because of their age.
A fundamentally different approach to avoid the problems associated with the systemic administration of a plasminogen activator to generate sufficient plasmin at the site of the thrombus, is to administer plasmin itself directly to the patient. This is because plasmin is ultimately the enzyme mediating thrombolysis initiated by plasminogen activators. Direct delivery of active plasmin directly into retracted thrombi would circumvent the inherent plasminogen deficiency of these thrombi and provide predictable, rapid and effective thrombolysis irrespective of plasminogen content.
Reich et al. in U.S. Pat. No: 5,288,489 discloses a fibrinolytic treatment that includes parenteral administration of plasmin into the body of a patient. The concentration and time of treatment were sufficient to allow active plasmin to attain a concentration at the site of an intravascular thrombus that is sufficient to lyse the thrombus or to reduce circulating fibrinogen levels. Reich et al., however, require generation of the plasmin from plasminogen immediately prior to its introduction into the body.
In contrast, Jenson in U.S. Pat. No. 3,950,513 discloses a porcine plasmin preparation that is asserted to be stabilized at low pH. The plasmin solution is neutralized before systemic administration to humans for thrombolytic therapy.
Yago et al. in U.S. Pat. No. 5,879,923 discloses plasmin compositions useful as a diagnostic reagent. The compositions of Yago et al. consist of low concentrations of plasmin at a neutral pH and an additional component that may be 1) an oligopeptide comprising at least two amino acids, or 2) at least two amino acids, or 3) a single amino acid and a polyhydric alcohol.
Plasmin represents a second mechanistic class of thrombolytic agents, distinct from the class of plasminogen activators. Although plasmin had been investigated as a potential thrombolytic agent, numerous technical difficulties have prevented effective clinical use of this fibrinolytic enzyme. These difficulties included the challenge of preparing pure plasmin that is free of all functional traces of the plasminogen activator used to generate plasmin from the inactive precursor, plasminogen. The thrombolytic activity of these earlier plasmin preparations was eventually attributed to the presence of contaminating plasminogen activators rather than to plasmin itself. The contaminating plasminogen activators, however, also triggered systemic bleeding at sites other than at the targeted thrombus. An additional drawback of streptokinase used for plasmin preparations is that its presence in a preparation of plasmin often causes adverse immune responses including fever and anaphylactic shock.
The most important limitation to the clinical use of plasmin is that, as a serine protease with broad specificity, it is highly prone to autodegradation and loss of activity at physiological pH. This provides severe challenges to the production of high-quality stable plasmin formulation suitable for prolonged periods of storage prior to use, and to safe and effective administration of plasmin to human patients suffering from occlusive thrombi.
What is needed, therefore, is a method of administering a stable form of active plasmin that is substantially free of plasminogen activators and which is active upon encountering a targeted vascular thrombotic occlusion.
What is also needed is a method of lysis of thrombi that directly delivers an activatable form of active plasmin via a catheter into the interior of the thrombus.
What is further needed is a method of thrombolysis in which an administered thrombolytic agent is restricted to the thrombotic site and which exhibits reduced systemic, and especially intracranial, hemorrhage.
Additionally, an improved method of dissolving thrombi forming occlusions in artificial devices such as hemodialysis grafts is also needed.
These and other objectives and advantages of the invention will become fully apparent from the description and claims that follow or may be learned by the practice of the invention.